Thermal and radiation damage to frozen hydrated specimens

Abstract

Although low temperature fixation seems to be superior to chemical fixation with respect to structure preservation, complex interactions between the electron beam and frozen hydrated specimens can limit its applications. A review is given of the present knowledge of beam heating and radiation damage with emphasis on recent developments in the field.Beam heating is found not to be a major limitation to cold stage microscopy. But radiolysis of ice and the formation of free radicals that subsequently attack organic matter in contact with ice can severely alter the specimen structure before a useful image is recorded. Use of low dose techniques may help in some cases, but the problem cannot be overcome in X-ray microanalysis of very small regions, when high beam doses must be used. The use of liquid helium stages operating near 4 K may reduce damage also in frozen hydrated specimens.Cold stage electron microscopy involves several difficult technical problems : preparation of fast frozen specimens with good preservation of the original structure, suitable for either transmission or scanning electron microscopy ; successful transfer of the frozen specimens into the electron microscope without excessive heating or deposition of frost, and finally keeping the specimen in the microscope at a very low temperature without condensation of contaminants from the microscope vacuum. In addition to these challenging problems the low temperature microscopist is faced with the complicated phenomena of electron beam sample interaction. There are two sides to this problem: generation of heat in the sample-'beam heating', and chemical and physical changes in the sample-'radiation damage'.Of the possible primary interactions between the electron beam and the sample the most important for light elements such as H, C, 0, N, the main constituents of biological specimens, are excitation of an atom or a collective excitation of several atoms, and atom ionization. The probability for displacement of a light atom, especially at the most often used acceleration voltage range of 25-100 keV, is very small. These primary interactions will be followed by secondary effects such as X-ray emission, light emission, temperature rise, electrostatic charging, bond scission or cross-linking, and mass loss (Cosslett, 1978). The curve of intensity v. energyloss given by Cosslett (1978) for carbon and reproduced in Fig. 1 shows the two main peaks of energy loss. The first, 0-10 eV, corresponds to UV absorption spectrum and probably contributes little to radiation damage. Most of the damage is caused by the second peak around 24 eV. This peak is partly due to collective excitations that may lead to heating of the sample, and to individual electron excitations and ionization which are probably the main source of damage. Although most of the energy transferred by the beam to the specimen will eventually be reduced to heat, heating itself is not the reason for radiation damage (Howie, 1980).

Imaging Beam‐Sensitive Materials by Electron Microscopy

“…In the second aspect, low‐temperature imaging minimizes the radiolytic and heating damage for beam‐sensitive materials, so as to increase their critical doses 212. Typically, the thermal vibrations required to achieve the energy–momentum conversion for electronic excitations are quenched by low temperature 64,65.…”

Cryosectioning of plant material frozen at high pressure

“…Interaction of electrons with the specimen leads to beam damage during cryosection analysis in the microscope. T h e mechanisms are complex and still far from being understood (Glaeser & Taylor, 1978;Talmon et al, 1979Talmon et al, , 1986Talmon, 1982Talmon, , 1987Dubochet et al, 1982a, b). Theoretical calculations (Talmon & Thomas, 1977) show that sample heating itself is negligible when specimens are kept at very low temperature.…”

Low‐temperature scanning electron microscopy of frozen hydrated mouse lung

“…Preliminary studies indicate that mouse lung shrinks 7-10% in linear dimension with overnight drying at 5-10 X lo4 Pa [28]. This is less than half the shrinkage that occurs with critical-point drying of lung and is comparable to the shrinkage with freeze-drying observed in other tissues [29]. The LTSEM with a temperature-controlled stage is an showing a dendritic pattern that we hypothesize is dendritic ice formed in the aqueous subphase between surfactant and epithelium.…”